The invention relates to the field of microbiology, in particular to microbiological analysis of fluid samples. In general, such analysis is aimed at determining the presence/absence of specific organic microobjects in the sample, at quantifying their number or concentration, and in some cases at identifying an unknown microorganism to various levels of detail. The term “organic microobject” may refer to any object of a large variety of microscopic objects of biological origin, in particular microorganisms and cells. Microorganisms include bacteria, fungi, archaea, protists, green algae, animals such as plankton, the planarian and the amoeba. Cells include, apart from bacteria, plant cells and mammalian cells, e.g. blood cells and tissue cells. Besides clinical diagnostics, such as testing of sputum or other bodily fluids, microbiological analysis has important industrial applications, for example in the food and beverage, pharmaceutical, personal care products, and environmental sectors. Current standard methods of testing are based on cell culturing and have time to results of days to weeks depending on the type of sample and microorganism. There is a need for microbiological analysis with increased throughput.
An example of such a rapid method is the one proposed by AES Chemunex (www.aeschemunex.com). Their FDA-approved ScanRDI-system provides a throughput from sample to result of 90 minutes and performs the analysis by laser scanning cytometry of filtered products. The steps of this method are: filtering the fluid sample, staining the possibly present microbiological contaminants with a fluorescent dye, optically scanning the surface of the filter with a large laser spot (5-10 μm) for detecting the possibly present microbiological contaminants, and imaging the areas surrounding the contaminants with a high-resolution (0.5 μm) microscope with an automated stage. The technique has been described in J. -L. Drocourt, P. Desfetes, J. Sanghera, Apparatus and process for rapid and ultrasensitive detection and counting of microorganisms by fluorescence, EP 0713087 B1 (1994).
An improved filter technology is provided by fluXXion (www.fluxxion.com). The technology is based on lithographically defined microsieves, which have a single well-defined pore size (down to 0.2 μm), are optically flat (which is advantageous from the point of view of the subsequent optical scanning step and also results in reduced backscattering) and thin so as to offer a low flow resistance and hence a higher filtration throughput compared to conventional membrane filters made from porous materials such as cellulose, nylon, polyvinyl chloride, polysulfone, polycarbonate, and polyester. An alternative to the two steps of low resolution imaging (via laser spot scanning) and high resolution imaging (with a microscope) is imaging the whole filter area at high resolution. In order to have a reasonable throughput for filtering it turns out that the filter area is much larger than the field of view of standard microscope objectives with the required resolution. For example, a resolution of 0.5 μm typically requires a 40X/NA0.65 objective lens with a field of view with a diameter of 0.5 mm. Typical filters have a diameter of several mm, so about an order of magnitude larger than the microscope objective lens. Clearly, this requires scanning the filter area, which is time-consuming and needs complex mechanics with high accuracy.
In a related context, rapid detection of pathogens and testing for antibiotic resistance/susceptibility can be crucial for proper treatment of patients with an infectious disease. Classical culturing techniques with patient samples typically take several days from sampling to end-result and have to be performed in large central microbiology labs. During this time, the patient can usually not remain untreated without suffering from severe consequences, which limits the medical practitioners to a guessing game using a broad antibiotic spectrum. This is not only economically costly but also increases the problems of antibiotic-resistive bacterial strains in hospital environments.
Speed can be gained and costs may be reduced by using automated and integrated tests on microfluidic devices. Speed may further be gained and costs may further be reduced by reducing the size of micro fluidic devices, to implement small reagent volumes and enable use of single-use plastic cartridges. A microfluidic system generally comprises a fluidic system into which the sample can be injected. It may further contain means for enriching bacteria and for separating them from human cells. The human cells may be examined separately in ways that would otherwise interfere with the bacterial analysis. Some microfluidic devices are adapted for screening mammalian cells for the presence of viral genes. Such devices may be capable of detecting all known types of microscopic pathogens. Microfluidic techniques have a great potential for rapid diagnosis of infectious diseases, for example by looking at a gene profile with e.g. real-time PCR, or through culturing in the microfluidic device and analysis of single bacterial divisions. There are various micro fluidic techniques for separating bacteria from human cells.
Examples include electrophoresis (see A. K. Balasubramanian, K. A. Soni, et al., A microfluidic device for continuous capture and concentration of microorganisms from potable water, Lab on a Chip, vol. 7, pp. 1315-1321, 2007), dielectrophoresis (see L. J. Yang, P. P. Banada, et al., A multifunctional micro-fluidic system for dielectrophoretic concentration coupled with immuno-capture of low numbers of listeria monocytogenes, Lab on a Chip, vol. 6, pp. 896-905, 2006) and magnetic bead separation (see Y. K. Cho, J. G. Lee, et al., One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device, Lab on a Chip, vol. 7, pp. 565-573, 2007).
However, a microfluidic device generally has a very limited sample throughput, typically on the order of a few μl/min. For that reason, there is a severe mismatch with real patient samples, such as from an oral or nasal swab, which often contain only 100 or less of the relevant bacteria in e.g. 1 ml of liquid, or blood samples containing several ml of liquid (typically 5-10 ml) and only a few free floating bacteria. Typically, real patient samples contain only a total of 10 to 100 of the relevant bacteria in 1 to 5 ml of sample liquid. Using only a fraction of the sample is therefore not an option. However, a ml volume does usually not fit into a microfluidic device. A connection between a macrofluidic sample volume and a microfluidic system is therefore required.
In addition to the above mentioned microfluidic techniques for separating bacteria and human cells in μl volumes, there are also many ways of doing this on the laboratory bench. A simple approach is to use syringe filters or centrifuge filters from e.g. Sartorius Stedim (www.sartorius-stedim.com), Whatman (www.whatman.com) or Millipore (www.millipore.com). Large pore-sizes capture the human cells while smaller pores catch the pathogens.
Wiegum and co-workers (S. E. Weigum, P. N. Floriano, et al., Cell-based sensor for analysis of EGFR biomarker expression in oral cancer, Lab. on a Chip, vol. 7, pp. 995-1003, 2007) have integrated filters in a PMMA cartridge and filtered out cells, which are then analyzed on a membrane. Here, the flow speeds are quite high, 250 μl/min-750 μl/min, but the cells, which are captured on the filter membrane, are not transferred to another liquid volume for further analysis.
Wu and Kado have used filters to enrich bacterial DNA in a sample (S. J. Wu and C. I. Kado, Preparation of milk samples for PCR analysis using a rapid filtration technique, Journal of Applied Microbiology, vol. 96, pp. 1342-1346, 2004). The authors use a filter with a 0.4 μl tm pore size to capture bacteria from a sample of milk. The membrane with the bacteria is then treated with a lysis buffer and the DNA is subsequently used for PCR. The very simple technique has a sensitivity of about 10 colony forming units (CFU) per ml of milk. For DNA analysis, there are also special centrifugation filters from SIRS-Lab (www.sirs-lab.com) which separate bacterial and human DNA. However, although efficient on the bench, none of the techniques above have been directly connected to a microfluidic chip.
Other methods of analysis include fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS), where the bacteria are labeled with antibodies for a specific strain and then sorted out. FACS machines can have a reasonably high throughput but are large and complex instruments and require specific labeling with antibodies. Magnetic separation of either the labeled cells or DNA bound to e.g. silica magnetic beads can be used to transfer the pathogen or pathogen DNA to a much smaller volume in a funnel structure. Nevertheless, the incubation of beads and sample still has to occur in a large volume, which is time consuming. Also, the transfer of the magnetic beads themselves, bound to cells or DNA, may also be problematic within the microfluidic system.
It is an object of the invention to reduce the total time that is required for imaging organic microobjects that are initially contained in a sample fluid.